A MODEL FOR THE KINETICS OF NEUTRAL AND ANIONIC DIPEPTIDE-PROTON COTRANSPORT BY THE APICAL MEMBRANE OF RAT-KIDNEY CORTEX

1. Kinetics of influx (mediated through peptide-proton cotransport) of two labelled dipeptides has been studied in apical membrane vesicles isolated from rat renal cortex. The substrates (neutral D-Phe-L-Ala an d anionic D-Phe-L-Glu) have previously been shown to be transported th rough a single system but with different stoichiometry of proton coupl ing. 2. The initial rate of influx of both peptides was determined und er a set of defined conditions allowing extravesicular pH, intravesicu lar pH, transmembrane pH and membrane potential (E(m)) to be varied sy stemically and independently. From this data the kinetic constants K-m and V-max were derived for each condition. Very substantial effects o f pH, pH gradient and membrane potential were found; there were consis tent quantitative differences when the substrates were compared. 3. Ef flux of the two peptides from preloaded vesicles was also determined. At pH 5.5 (intra- and extravesicular), but not at pH 7.4, the rate con stants for efflux of the two peptides were similar and addition to the extravesicular medium of unlabelled D-Phe-L-Glu (but not D-Phe-L-Ala) trans-stimulated efflux of both peptides to a similar extent; the ext ent of this trans-stimulation was insensitive to alterations in membra ne potential. 4. A model based on a combination of classical carrier t heory (the carrier being negatively charged) and of two sequential pro tonation steps (both to external sites predicted to be in the membrane electrical field) is described. Qualitatively this adequately account s for all the observations made and allows for the dependence of the s toichiometry of proton-peptide coupling on the net charge carried by t he substrate. Quantitatively a 50-fold greater rate of reorientation o f the free carrier when unprotonated is predicted to be responsible fo r the coupling of proton and peptide transport. 5. Our results and the model are discussed with respect to the recently elucidated primary s tructure of mammalian peptide transporters.